Abstract: Abstract: Objective　To extract cortical neurons of newborn SD rats within 24 h by papain combined with DNA enzyme, and to improve the primary culture method in vitro. Methods　The cerebral cortex of newborn SD rats was separated from the cerebral vascular membrane within 24 h. After sequentially digested and purified by papain and DNA enzyme, the neurons were inoculated with DMEM-F12 medium containing 10% fetal bovine serum. After 4 hours, it was replaced with pre-warmed Neurobasal-A neuronal medium containing B27, and cultured continuously for 7 days. 1×105, 5×105 and 1×106 cells/mL were seeded into 6-well plates soaked in L-polylysine, respectively and the growth state of the cells was observed under a microscope. The neurons cultured for 7 days were identified by immunofluorescence method of β -Tubulin and immunohistochemical method of neuron specific nuclear protein (Neun) antibody. The purity of neurons was identified by immunofluorescence method of neuron microtubule-associated protein 2 antibody (MAP2). Results　The most suitable inoculation method was 5×105 cells/mL/well. The cortical neurons partially adhered to the wall after 24-h inoculation. Three days after inoculation, adherent cells gradually increased, synapses further grew and extended, and cross-linked into sparse networks. Seven days after inoculation, the neurons still grew in large numbers, the cell body was plump, and a tighter neural network system was formed. The cells were identified by immunofluorescence method of β-Tubulin and immunohistochemical method of Neun antibody, and the neuron purity was (91.06±1.51) % by immunofluorescence method of neuron marker MAP2. Conclusion　Newborn SD rats within 24 hours are used to separate the cerebral cortex, and after digestion with papain and DNA enzyme, high-quality cortical neurons can be extracted.

Key words: neurons, primary cell culture, papain, deoxyribonucleases, rats, Sprague-Dawley, neonatal SD rats, cell model